Human rotaviruses (HRVs) are the major cause of acute diarrhea in infants and young children. Here, a real-time reverse transcription polymerase chain reaction assay targeting the rotaviral VP4 gene (VP4-RT-qPCR) was established to evaluate the inactivation of HRV upon chlorine disinfection, based on a previous report that damage to the 1227-2354bp region of the VP4 gene was associated with eliminated HRV infectivity by chlorine. In this study, inactivation of HRV by 0.6mg/L free chlorine was assessed in phosphate buffered saline (PBS; pH 7.2), and tap and river water samples, using both TCID(50) and RT-qPCR (VP2- and VP4-RT-qPCR) assays, respectively. Among the samples tested, the VP2-RT-qPCR method did not show significant inactivation after chlorine disinfection; however, the reduction in VP4-RT-qPCR signal was correlated with decreased HRV infectivity. Moreover, the higher sensitivity of the VP4-RT-qPCR assay allowed for assessment of chlorine HRV inactivation at longer exposure times compared with the conventional TCID(50) assay. Collectively, these results indicated that the VP4-RT-qPCR assay is a rapid, sensitive, and reliable tool to detect infectious HRV following chlorine inactivation, and highlights the potential for further development of qPCR/RT-qPCR assays to provide information regarding viral infectivity from drinking water plants.